During FY 2013, we performed laboratory experiments based on previous observations made during our field work in Montana and California. Our investigations of tick-borne relapsing fever in western Montana produced our finding of both genomic groups of Borrelia hermsii in ticks and small mammals at Flathead Lake. Also, one patient that was infected on Wild Horse Island was dually infected with Borrelia hermsii spirochetes belonging to different genomic groups. We also found that two patients that had slept together in the same bed were each infected with spirochetes belonging to different genomic groups. Therefore, we became quite interested in determining how spirochetes in genetically different clones were maintained at one endemic focus. Our experiments investigated the ability of individual Ornithodoros hermsi ticks to become dually or super-infected with different genetic strains of spirochetes. To test the vector competence of Ornithodoros hermsi with distinct Borrelia hermsii genotypes, we sequentially infected ticks with genetically different spirochetes, examined the ticks for persistent infection, and tested them for transmission to mice. We found that ticks infected with one genetic type of Borrelia hermsii remained susceptible to infection to a different genotype acquired during subsequent feedings. The ticks became persistently infected with both types of spirochetes and transmitted spirochetes of one or both types. These results suggest that ticks in an endemic focus may be able to become dually infected and co-transmit multiple genetic types of Borrelia hermsii during a single feeding ( Policastro, P.F., Raffel, S.J., and Schwan, T.G. Borrelia hermsii acquisition order in superinfected ticks determines transmission efficiency. Infect. Immun. 81: 2899-2908, 2013). In another series of experiments, we investigated genetically altered Borrelia hermsii that had the expression locus for antigenic variation inactivated. We used novel genetic constructs to make the spirochetes unable to switch their bloodstream surface proteins, which rendered the spirochetes attenuated and unable to cause relapses in mice. Experimental infections in SCID mice demonstrated that in the absence of antibody, the spirochetes that were unable to switch did persist in high cell densities. Also this last year we characterized an isolate of the relapsing fever spirochete Borrrelia hermsii we cultured from a clinically ill domestic dog. The dog was infected in central Washington in a region endemic for human relapsing fever. We applied multi-locus sequence typing and found that the spirochete belonged to Genomic Group II, and was most similar to B. hermsii we have isolated in past years from human patients infected in western Montana and northern Idaho. This is the first case report and isolation of the Borrelia hermsii from a dog. (Kelly, A.L., Raffel, S.J., Fischer. R.J., Bellinghausen, M., Stevenson, C., Schwan, T.G. First isolation of the relapsing fever spirochete, Borrelia hermsii, from a domestic dog. Ticks and Tick-Borne Diseases (revised manuscript submitted July 16, 2013). The PI retired on January 10, 2014.